Pre-Analytical and Analytical Variables of Label-Independent Enrichment and Automated Detection of Circulating Tumor Cells in Cancer Patients

Koch, Claudia; Joosse, Simon A.; Schneegans, Svenja; Wilken, Okka J. W.; Janning, Melanie; Loreth, Desirée; Müller, Volkmar; Banys-Paluchowski, Malgorzata; Horst, Ludwig J; Loges, Sonja; Peine, Sven; Wikman, Harriet; Gorges, Tobias M.; Pantel, Klaus

doi:10.3390/cancers12020442

Cited by 29 publications

(36 citation statements)

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“…For automatic cell identification, the Xcyto Quantitative Cell Imager (Chemometec) was used. 27 Histology and micro–computed tomography (µCT) analyses of the spines and the right femora were performed as described in Supplementary Material.…”

Section: Methodsmentioning

confidence: 99%

ALCAM contributes to brain metastasis formation in non-small-cell lung cancer through interaction with the vascular endothelium

et al. 2020

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Abstract Background Brain metastasis (BM) in non-small-cell lung cancer (NSCLC) has a very poor prognosis. Recent studies have demonstrated the importance of cell adhesion molecules in tumor metastasis. The aim of our study was to investigate the role of activated leukocyte cell adhesion molecule (ALCAM) in BM formation in NSCLC. Methods Immunohistochemical analysis was performed on 143 NSCLC primary tumors and BM. A correlation between clinicopathological parameters and survival was developed. Biological properties of ALCAM were assessed in vitro by gene ablation using CRISPR/Cas9 technology in the NCI-H460 NSCLC cell line and in vivo by intracranial and intracardial cell injection of NCI-H460 cells in NMRI-Foxn1nu/nu mice. Results ALCAM expression was significantly upregulated in NSCLC brain metastasis (P = 0.023) with a de novo expression of ALCAM in 31.2% of BM. Moderate/strong ALCAM expression in both primary NSCLC and brain metastasis was associated with shortened survival. Functional analysis of an ALCAM knock-out (KO) cell line showed a significantly decreased cell adhesion capacity to human brain endothelial cells by 38% (P = 0.045). In vivo studies showed significantly lower tumor cell dissemination in mice injected with ALCAM-KO cells in both mouse models, and both the number and size of BM were significantly diminished in ALCAM depleted tumors. Conclusions Our findings suggest that elevated levels of ALCAM expression promote BM formation in NSCLC through increased tumor cell dissemination and interaction with the brain endothelial cells. Therefore, ALCAM could be targeted to reduce the occurrence of BM. Key Points 1. ALCAM expression associates with poor prognosis and brain metastasis in NSCLC. 2. ALCAM mediates interaction of NSCLC tumor cells with brain vascular endothelium. 3. ALCAM might represent a novel preventive target to reduce the occurrence of BM in NSCLC.

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Section: Methodsmentioning

confidence: 99%

ALCAM contributes to brain metastasis formation in non-small-cell lung cancer through interaction with the vascular endothelium

et al. 2020

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“…Blood was collected in parallel in three different blood collection tubes, that is, EDTA (BD Vacutainer, Franklin Lakes, NJ, US), Streck cell‐free circulating DNA (cfDNA) BCT® (Streck, La Vista, NE, US), and Transfix (Cytomark, Buckingham, UK; 7.5 mL each). EDTA was chosen due to its broad applicability, whereas Streck is commonly used in ctDNA analyses and we have recently shown the suitability of Transfix for CTC analysis (Koch et al , 2020). Plasma removal without hemolysis was not possible from Transfix tubes, so these tubes were only used for CTC analyses.…”

Section: Methodsmentioning

confidence: 99%

Pre‐analytical factors affecting the establishment of a single tube assay for multiparameter liquid biopsy detection in melanoma patients

et al. 2020

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The combination of liquid biomarkers from a single blood tube can provide more comprehensive information on tumor development and progression in cancer patients compared to single analysis. Here, we evaluated whether a combined analysis of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and circulating cell-free microRNA (miRNA) in total plasma and extracellular vesicles (EV) from the same blood sample is feasible and how the results are influenced by the choice of different blood tubes. Peripheral blood from 20 stage IV melanoma patients and five healthy donors (HD) was collected in EDTA, Streck, and Transfix tubes. Peripheral blood mononuclear cell fraction was used for CTC analysis, whereas plasma and EV fractions were used for ctDNA mutation and miRNA analysis. Mutations in cell-free circulating DNA were detected in 67% of patients, with no significant difference between the tubes. CTC was detected in only EDTA blood and only in 15% of patients. miRNA NGS (nextgeneration sequencing) results were highly influenced by the collection tubes and could only be performed from EDTA and Streck tubes due to hemolysis in Transfix tubes. No overlap of significantly differentially expressed miRNA (patients versus HD) could be found between the tubes in total plasma, whereas eight miRNA were commonly differentially regulated in the EV fraction. In summary, high-quality CTCs, ctDNA, and miRNA data from a single blood tube can be obtained.

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“…Immunocytochemical detection of nuclear ARv7 expression on CTCs has been developed into a new predictive biomarker (131). Nevertheless, a marked intrapatient heterogeneity in ARv7 expression between individual CTCs has been reported by single-cell analysis (38), which may also affect clinical outcome. Besides transcriptional plasticity, amplifications of the AR gene locus could be detected in 30% to 38% of patients with CRPC (132,133), and AR mutations were identified in CTC-enriched peripheral blood samples from patients with CRPC (134).…”

Section: Prostate Cancermentioning

confidence: 99%

“…Moreover, new devices for the in vivo detection and/or capture of CTCs (35)(36)(37) have opened a new avenue for CTC research, which may overcome the limitation of low CTC counts present in a usual 7.5-mL blood tube. Besides the FDA-cleared CELLSEARCH system that uses EPCAM-coated magnetic particles for CTC enrichment, these new devices include, for example, assays for label-independent size-based enrichment (38) and photoacoustic detection (35), as well as a temporary indwelling intravascular aphaeretic system (36) and an EPCAM-coated in vivo capture wire (37).…”

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confidence: 99%

Liquid Biopsy: From Discovery to Clinical Application

2021

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The liquid biopsy concept was introduced for circulating tumor cells (CTC) 10 years ago (1) and rapidly extended to circulating tumor DNA (ctDNA; ref. 2) and other tumorderived products such as circulating cell-free RNA (noncoding and messenger RNA; ref.3), extracellular vesicles (4), or tumor-educated platelets (ref. 5; Fig. 1). Research on the two key components of liquid biopsy assays, CTCs and ctDNA, is a very active field, with more than 26,070 publications listed under the key phrase "CTC" and more than 5,720 for "ctDNA" in PubMed in September 2020 (i.e., on average 30 to 40 new publications each week for CTCs in 2020). These liquid biomarkers are used in more than 557 clinical trials registered at the NCI website (http://clinicaltrials.gov; 325 for CTCs and 232 for ctDNA; among them, 7 involving both biomarkers). Strong evidence for CTCs and ctDNA as prog-absTRaCT Over the past 10 years, circulating tumor cells (CTC) and circulating tumor DNA (ctDNA) have received enormous attention as new biomarkers and subjects of translational research. Although both biomarkers are already used in numerous clinical trials, their clinical utility is still under investigation with promising first results. Clinical applications include early cancer detection, improved cancer staging, early detection of relapse, real-time monitoring of therapeutic efficacy, and detection of therapeutic targets and resistance mechanisms. Here, we propose a conceptual framework of CTC and ctDNA assays and point out current challenges of CTC and ctDNA research, which might structure this dynamic field of translational cancer research.Significance: The analysis of blood for CTCs or cell-free nucleic acids called "liquid biopsy" has opened new avenues for cancer diagnostics, including early detection of tumors, improved risk assessment and staging, as well as early detection of relapse and monitoring of tumor evolution in the context of cancer therapies. TeChNOlOgies fOR CTC aND ctDNa DeTeCTiONSeveral recent articles have reviewed the technologies used for CTC and ctDNA analyses (13-15). We will therefore only briefly describe the principles of CTC and ctDNA assays (Fig. 3). CTCsVarious devices have been developed to enrich and detect CTCs, with a focus on devices able to select and detect CTCs that underwent epithelial-mesenchymal transition (EMT) and lack expression of EPCAM as the most frequently used cell surface protein used for CTC enrichment of blood from Research.

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Pre-Analytical and Analytical Variables of Label-Independent Enrichment and Automated Detection of Circulating Tumor Cells in Cancer Patients

Cited by 29 publications

References 36 publications

ALCAM contributes to brain metastasis formation in non-small-cell lung cancer through interaction with the vascular endothelium

ALCAM contributes to brain metastasis formation in non-small-cell lung cancer through interaction with the vascular endothelium

Pre‐analytical factors affecting the establishment of a single tube assay for multiparameter liquid biopsy detection in melanoma patients

Liquid Biopsy: From Discovery to Clinical Application

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